Methods For Preventing, Removing, Reducing, or Disrupting Biofilm

ABSTRACT

The present invention relates to methods for preventing, removing, reducing, or disrupting biofilm present on a surface, comprising contacting the surface with an alpha-amylase derived from a bacterium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional applicationentitled “Methods for preventing, removing, reducing, or disruptingbiofilm”, filed on Sep. 8, 2004 (Ser. No. 60/608,535) which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to improved methods of preventing,removing, reducing, or disrupting biofilm formation on a surface.

DESCRIPTION OF THE RELATED ART

Biofilms are biological films that develop and persist at the surfacesof biotic or abiotic objects in aqueous environments from the adsorptionof microbial cells onto the solid surfaces. This adsorption can providea competitive advantage for the microorganisms since they can reproduce,are accessible to a wider variety of nutrients and oxygen conditions,are not washed away, and are less sensitive to antimicrobial agents. Theformation of the biofilm is also accompanied by the production ofexo-polymeric materials (polysaccharides, polyuronic acids, alginates,glycoproteins, and proteins) which together with the cells form thicklayers of differentiated structures separated by water-filled spaces.The resident microorganisms may be individual species of microbial cellsor mixed communities of microbial cells, which may include aerobic andanaerobic bacteria, algae, protozoa, and fungi. Thus, the biofilm is acomplex assembly of living microorganisms embedded in an organicstructure composed of one or more matrix polymers which are secreted bythe resident microorganisms.

Biofilms can develop into macroscopic structures several millimeters orcentimeters in thickness and cover large surface areas. These formationscan play a role in restricting or entirely blocking flow in plumbingsystems, decreasing heat transfer in heat exchangers, or causingpathogenic problems in municipal water supplies, food processing,medical devices (e.g., catheters, orthopedic devices, implants,endoscopes). Moreover, biofilms often decrease the life of materialsthrough corrosive action mediated by the embedded microorganisms. Thisbiological fouling is a serious economic problem in industrial waterprocess systems, pulp and paper production processes, cooling watersystems, injection wells for oil recovery, cooling towers, porous media(sand and soil), marine environments, and air conditioning systems, andany closed water recirculation system. Biofilms are also a severeproblem in medical science and industry causing dental plaque,infections (Costerton et al., 1999, Science 284:1318-1322), contaminatedendoscopes and contact lenses, prosthetic device colonisation andbiofilm formation on medical implants.

The removal or prevention of biofilm traditionally requires the use ofdispersants, surfactants, detergents, enzyme formulations,anti-microbials, biocides, boil-out procedures, and/or corrosivechemicals, e.g., base. Procedures for using these measures are wellknown in the art. For example, removal of biofilm built-up in a papermachine in the pulp and paper industry traditionally requires a depositcontrol program including proper housekeeping to keep surfaces free ofsplashed stock, anti-microbial treatment of fresh water and additives,the use of biocides to reduce microbiological growth on the machine, andscheduled boil-outs to remove the deposits that do form.

Bacteria growing in biofilms are more resistant to antibiotics anddisinfectants than planktonic cells and the resistance increases withthe age of the biofilm. Bacterial biofilm also exhibits increasedphysical resistance towards desiccation, extreme temperatures or light.As mentioned, biofilm formation causes industrial, environmental andmedical problems and the difficulties in cleaning and disinfection ofbacterial biofilm with chemicals is a major concern in many industries.Furthermore, the trend towards milder disinfection and cleaningcompositions to reduce their environmental impact may increase theinsufficient cleaning of surfaces covered with biofilm.

It is an object of the present invention to provide improved methods forpreventing or removing biofilm present on a surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the percentage (%) of total hydrolysis ofraw wheat starch for three different alpha-amylases.

FIG. 2 is a chromatogram comparing 1) Alpha-amylase A, 2) detergentalone 3) Alpha-amylase C biofilm removal.

SUMMARY OF THE INVENTION

The present invention relates to methods for preventing, removing,reducing, or disrupt biofilm formation on a surface, comprisingcontacting the surface with alpha-amylase derived from a bacterium.

The term “surface” is defined herein as any surface which may be coveredby biofilm or is prone to biofilm formation. Examples of surfaces may beany hard surface such as metal, plastics, rubber, board, glass, wood,paper, concrete, rock, marble, gypsum and ceramic materials, whichoptionally are coated, for example, with paint or enamel; any softsurface such as fibers of any kind (e.g., yarns, textiles, vegetablefibers, rock wool, and hair); or any porous surfaces; skin (human oranimal); keratinous materials (e.g., nails); and internal organs (e.g.,lungs). The hard surface can be present as a part of a cooling tower,water treatment plant, water tanks, dairy, food processing plant,chemical or pharmaceutical process plant, or medical device (e.g.,catheters, orthopedic devices, implants). The porous surface can bepresent in a filter, e.g., a membrane filter.

The term “effective amount” is defined herein as the amount of one ormore alpha-amylases that is sufficient to degrade a microbially-producedbiofilm comprising alpha-1,4 glucosidic linkages. The effective amountof the one or more alpha-amylase will depend on factors including: thealpha-amylase(s) in question, whether the aim is preventing, removing,or reducing biofilms present on a surface, the period of time desirablefor, e.g., degrading a microbially-produced biofilm. Highamounts/concentrations of enzyme(s) will in general require shortertimes of treatment, while low amounts/concentrations longer times.Further, for instance, preventing biofilm on a surface prone to biofilmformation will in general require lower amounts/concentrations ofenzyme(s) than the actual removal of biofilm from a correspondingcontaminated surface. However, typical effective usage levels arebetween 0.005 to 500 mg of alpha-amylase protein per L biofilm controlsolution, preferably between 0.01-100 mg of enzyme protein per L biofilmcontrol solution. The term “biofilm control solution” refers to asolution used according to the invention for preventing, removing,reducing or disrupting biofilm present on a surface. The method of theinvention may result in 10-10⁸-fold, preferably 10³-10⁶-fold biofilmreduction in terms of average plate count under the conditions indicatedin Example 4 below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved methods of preventing,removing, or reducing biofilms present on a surface, comprisingcontacting the surface with an effective amount of an alpha-amylase asdefined below. The methods of the present invention may be used toprevent, remove, reduce, or disrupt biofilm formation on a surface. Oneof ordinary skill in the art will recognize that such methods may beemployed at different stages of biofim formation.

By using an alpha-amylase in an effective amount of surfaces improvedbiofilm prevention and/or removal is obtained, especially in thoseinstances where some of the microbes present in the biofilm producealpha-1,4 linked glucose polysaccharides such as amylose, amylopectin,mixtures of these two polysaccharides (i.e., starch), and glycogen.

In the first aspect the invention relates to a method for preventing orremoving biofilm on a surface, comprising contacting the surface with analpha-amylase derived from a bacterium. In a preferred embodiment thebacterial alpha-amylase is derived from a strain of Bacillus.

Alpha-Amylase

The alpha-amylase used according to the invention is derived from abacterium, preferably derived from a strain of Bacillus sp., especiallyselected from the group consisting of: the AA560 alpha-amylase disclosedas SEQ ID NO: 2 in WO 00/60060 (SEQ ID NO: 2 herein), the Bacillusflavothermus disclosed in U.S. patent application Ser. No. 10/877,847,Bacillus sp. alpha-amylases disclosed in WO 95/26397, alpha-amylasesderived from Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375,DSMZ no. 12649, KSM AP1378 (WO 97/00324), KSM K36 or KSM K38 (EP1,022,334), and the #707 alpha-amylase disclosed by Tsukamoto et al.,Biochemical and Biophysical Research Communications, 151 (1988), pp.25-31.

In a preferred specific embodiment of the invention the alpha-amylase isthe AA560 alpha-amylase shown in SEQ ID NO: 2 herein and/or the AMY1048alpha-amylase shown in SEQ ID NO: 4 herein, or an alpha-amylase having adegree of identity of at least 60%, preferably at least 70%, morepreferred at least 80%, even more preferred at least 90%, such as atleast 95%, at least 96%, at least 97%, at least 98% or at least 99% toany of the sequences shown in SEQ ID NOS: 2 or 4 herein.

In a preferred embodiment the alpha-amylase used is a variant of theparent alpha-amylase disclosed in SEQ ID NO: 2 herein having a deletionin positions D183 and/or G184, preferably wherein said alpha-amylasevariant further has a substitution in or corresponding to positionN195F, especially wherein the parent alpha-amylase has one or more ofthe following deletions/substitutions in SEQ ID NO: 2 herein: Delta(R81-G182); Delta (D183-G184); Delta (D183-G184)+N195F;R181Q+N445Q+K446N; Delta (D183-G184)+R181Q, Delta (D183-G184) and one ormore of the following substitutions: R118K, N195F, R320K, R458K,especially wherein said parent alpha-amylase has the followingmutations:

Delta (D183+G184)+R118K+N195F+R320K+R458K (i.e., in SEQ ID NO: 2herein).

In another preferred embodiment the alpha-amylase is the AA560alpha-amylase shown in SEQ ID NO: 2 or a variant thereof furthercomprising one or more of the following substitutions: M9L, M202L,V214T, M323T, M382Y or M9L, M202L, V214T, M323T and E345R.

In a preferred embodiment the alpha-amylase has a percentage (%) ofhydrolyzed raw starch that is higher than 15, preferably 25, especially35, after 5 hours at 40° C., 3 mg enzyme protein per g starch, pH 8.0(See Example 2 and FIG. 1).

In another preferred embodiment the alpha-amylase comprisesAsn-Gly-Thr-Met-Met-Gln-Tyr-Phe-Glu-Trp in its N-terminal amino acidregion. Examples of such alpha-amylase include Alpha-Amylase A andAlpha-Amylase B used in Example 2.

In an embodiment the alpha-amylase is derived from a strain of Bacilluslicheniformis with the sequence shown as SEQ ID NO: 6 herein, or analpha-amylase having a degree of identity of at least 60%, preferably atleast 70%, more preferred at least 80%, even more preferred at least90%, such as at least 95%, at least 96%, at least 97%, at least 98% orat least 99% to any of the sequences shown in SEQ ID NO: 6 herein,preferably with a substitution in a position corresponding to positionM197, preferably M197L, T, I, N, D, Q, E,P,W, especially M197L or T.

Commercially available alpha-amylase products or products comprisingalpha amylases include product sold under the following trade names:STAINZYME™, DURAMYL™ (Novozymes A/S, Denmark), BIOAMYLASE D(G),BIOAMYLASE™ L (Biocon India Ltd.), KEMZYM™ AT 9000(Biozym Ges. m.b.H,Austria), PURASTAR™ ST, PURASTAR™ HPAmL, PURAFECT™ OxAm, RAPIDASE™ TEX(Genencor Int. Inc, USA), KAM (KAO, Japan).

In a preferred embodiment, surfaces prone to biofilm formation may besubjected to the methods of the present invention as a preventativemeasure prior to any biofilm formation so no biofilm forms.Alternatively, at the first indication of biofim formation, the methodsmay be used to prevent further formation and to remove the biofim thathas deposited on a surface. Furthermore, in situations where there is aheavy build-up of biofilm on a surface, the methods may be used toreduce the level of biofilm or to remove it partially or completely.

A biofilm may comprise an integrated community of one or two or moremicroorganisms or predominantly a specific microorganism (Palmer andWhite, 1997, Trends in Microbiology 5: 435440; Costerton et al., 1987,Annual Reviews of Microbiology 41: 435-464; Mueller, 1994, TAPPIProceedings, 1994 Biological Sciences Symposium 195-201). In the methodsof the present invention, the one or more microorganisms may be anymicroorganism involved in biofilm formation including, but not limitedto, aerobic bacteria or anaerobic bacteria (Gram positive and Gramnegative), fungi (yeast or filamentous fungus), algae, and/or protozoa.Contemplated bacteria include bacteria selected from the groupconsisting of. Pseudomonas spp. including Pseudomonas aeruginosa,Azotobacter vinelandii, Escherichia coli, Corynebacterium diphteriae,Clostridium botulinum, Streptococcus spp, Acetobacter, Leuconostoc,Betabacterium, Pneumococci, Mycobacterium tuberculosis, Aeromonas,Burkholderie, Flavobacterium, Salmonella, Staphylococcus.

In a preferred embodiment, the microorganism is an aerobic bacterium. Ina more preferred embodiment, the aerobic bacterium is an Aeromonasstrain. In another more preferred embodiment, the aerobic bacterium is aBurkholderie strain. In another more preferred embodiment, the aerobicbacterium is a Flavobacterium strain. In another more preferredembodiment, the aerobic bacterium is a Microbacterium strain. In anothermore preferred embodiment, the aerobic bacterium is a Pseudomonasstrain. In another more preferred embodiment, the aerobic bacterium is aSalmonella strain. In another more preferred embodiment, the aerobicbacterium is a Staphylococcus strain. In another more preferredembodiment, the aerobic bacterium is from the family Enterobacteriaceae(including e.g., Escherichia coli).

In a most preferred embodiment, the aerobic bacterium is Burkholderiecepacia. In another most preferred embodiment, the aerobic bacterium isa Microbacterium imperiale or Mycobacterium tuberculosis. In anothermost preferred embodiment, the aerobic bacterium is Pseudomonasaeruginosa. In another most preferred embodiment, the aerobic bacteriumis Pseudomonas fluorescens. In another most preferred embodiment, theaerobic bacterium is Pseudomonas oleovorans. In another most preferredembodiment, the aerobic bacterium is Pseudomonas pseudoalcaligenes. Inanother most preferred embodiment, the aerobic bacterium is Salmonellaenteritidis. In another most preferred embodiment, the aerobic bacteriumis Staphylococcus aureus. In another most preferred embodiment, theaerobic bacterium is Staphylococcus epidermidis.

In another preferred embodiment, the microorganism is an anaerobicbacteria. In another more preferred embodiment, the anaerobic bacteriumis a Desulfovibrio strain. In another most preferred embodiment, theanaerobic bacterium is Desulfovibrio desulfuricans.

In another preferred embodiment, the microorganism is a fungus such as ayeast or filamentous fungus. In another more preferred embodiment, theyeast is a Candida strain. In another most preferred embodiment, theyeast is Candida albicans.

As mentioned above the treatment time for preventing or removing biofilmwill depend on the dosage of the alpha-amylase, and the level of biofilmon the surface or prone to the area in question, but should preferablybe adapted to the time normally used for conventional treatment ofbiofilm with antibiotics, biocides, bactericides, fungicides, bleachingagents, surfactants, caustic, and/or biopolymer degrading agents.Consequently, the dosage of the alpha-amylase may be adjusted accordingto the time period used during conventional treatments. However, wherethe alpha-amylase treatment is a separate step in the processing, thedosage of the alpha-amylase used will depend on the time period desiredto accomplish the treatment.

In terms of alpha-amylase activity, the appropriate dosage ofalpha-amylase for preventing or removing biofilms will depend on theamount of biofilm on the surface or prone to the area in question. Theskilled person may determine a suitable alpha-amylase unit dosage. Thedosage may be expressed in alpha-amylase units. Alpha-amylase units maybe determined as “KNU”, using the assay described below in the“Materials & Methods”-section. Biofilm contaminated or prone areas arepreferably treated for between 1 minute and 2 days, preferably between10 minutes and 1 day, preferably between 1 hour and 15 hours, morepreferably less that 10 hours, with an alpha-amylase dosage of between0.005 to 500 mg of alpha-amylase protein per L biofilm control solution,preferably between 0.01 to 100 mg of alpha-amylase protein per L biofilmcontrol solution.

The alpha-amylase may be part of a composition to be used in the methodsof the present invention. The composition may be in any form suitablefor the use in question, e.g., in the form of a dry powder, agglomeratedpowder, or granulate, in particular a non-dusting granulate, liquid, inparticular a stabilized liquid, or protected alpha-amylase. Granulatesand agglomerated powders may be prepared by conventional methods, e.g.,by spraying the alpha-amylase onto a carrier in a fluid-bed granulator.The carrier may consist of particulate cores having a suitable particlesize. The carrier may be soluble or insoluble, e.g., a salt (such assodium chloride or sodium sulfate), sugar (such as sucrose or lactose),sugar alcohol (such as sorbitol), or starch. The alpha-amylase may becontained in slow-release formulations. Methods for preparingslow-release formulations are well known in the art. Liquidalpha-amylase preparations may, for instance, be stabilized by addingnutritionally acceptable stabilizers such as a sugar, sugar alcohol, oranother polyol, and/or lactic acid or another organic acid according toestablished methods.

The composition may be augmented with one or more agents for preventingor removing the formation of the biofilm. These agents may include, butare not limited to, dispersants, surfactants, detergents, other enzymes,anti-microbials, and biocides.

In a preferred embodiment, the agent is a surfactant. The surfactant maybe a non-ionic including semi-polar and/or anionic and/or cationicand/or zwitterionic surfactant.

Anionic surfactants contemplated include linear alkylbenzenesulfonate,alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcoholethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methylester, alkyl- or alkenylsuccinic acid or soap.

Non-ionic surfactants contemplated include alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The surfactants may be present at a level of from 0.1% to 60% by weightof the enzyme biofilm removal composition.

In a more preferred embodiment, the surfactant is sodium dodecylsulfate, quaternary ammonium compounds, alkyl pyridinium iodides, Tween80, Tween, 85, Triton X-100, Brij 56, biological surfactants,rhamnolipid, surfactin, visconsin, or sulfonates.

The formation of biofilm is generally accompanied by the production ofexo-polymeric materials (polysaccharides, polyuronic acids, alginates,glycoproteins, and proteins) which together with the cells form thicklayers of differentiated structures separated by water-filled spaces(McEldowney and Fletcher, 1986, Journal of General Microbiology 132:513-523; Sutherland, Surface Carbohydrates of the Prokaryotic Cell,Academic Press, New York, 1977, pp. 27-96). In the methods of thepresent invention, the alpha-amylase composition may further compriseone or more other enzymes capable of degrading the exo-polymericmaterials such as polysaccharides, polyuronic acids, alginates,glycoproteins, and proteins.

Other Enzyme Activities

In a preferred embodiment, the one or more other enzymes may be selectedfrom the group consisting of an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, oxididases, including carbohydrate oxidases,peroxidases, laccase, lipase, mannosidase, pectinolytic enzyme,peptidoglutaminase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase.

The other enzyme(s) may be selected according to the properties of thespecific biofilm which is to be removed, or a combination of severalenzymes having different enzyme activities may be used.

In a preferred embodiment, the other enzyme is selected from the groupconsisting of 1,2-1,3-alpha-D-mannan mannohydrolase, 1,3-beta-D-xylanxylanohydrolase, 1,3-beta-D-glucan glucanohydrolase, 1,3(1,3;1,4)-alpha-D-glucan 3-glucanohydrolase, 1,3(1,3;1,4)-beta-D-glucan3(4)-glucanohydrolase, 1,3-1,4-alpha-D-glucan 4-glucanohydrolase,1,4-alpha-D-glucan glucanehydrolase, 1,4-alpha-D-glucan glucohydrolase,1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, 1,4-beta-D-glucanglucohydrolase, 1,4-beta-D-xylan xylanohydrolase, 1,4-beta-D-mannanmannanohydrolase, 1,5-alpha-L-arabinan 1,5-alpha-L-arabinanohydrolase,1,4-alpha-D-glucan maltohydrolase, 1,6-alpha-D-glucan6-glucanohydrolase, 2,6-beta-D-fructan fructanohydrolase, alpha-Dextrin6-glucanohydrolase, alpha-D-galactoside galactohydrolase,alpha-D-glucoside glucohydrolase, alpha-D-mannoside mannohydrolase,acyineuraminyl hydrolase, Aerobacter-capsular-polysaccharidegalactohydrolase, beta-D-fructofuranoside fructohydrolase,beta-D-fucoside fucohydrolase, beta-D-fructan fructohydrolase,beta-D-galactoside galactohydrolase, beta-D-glucoside glucohydrolase,beta-D-glucuronoside, glucuronosohydrolase, beta-D-mannosidemannohydrolase, beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, cellulose-sulfate sulfohydrolase, collagenase, dextrin6-alpha-D-glucanohydrolase, glycoprotein-phosphatidylinositolphosphatidohydrolase, hyaluronate 4-glycanohydrolase,hyaluronoglucuronidase, pectin pectylhydrolase, peptidoglycanN-acetylmuramoylhydrolase, phosphatidylcholine 2-acylhydrolase,phosphatidylcholine 1-acylhydrolase, poly(1,4-alpha-D-galacturonide),poly(1,4-(N-acetyl-beta-D-glucosaminide))-glycanohydrolase, sucrosealpha-glucosidase, triacylglycerol acylhydrolase, and triacylglycerolprotein-acylhydrolase.

Proteolytic Enzyme

The other enzyme may be any enzyme having proteolytic activity under theactual process conditions. Thus, the enzyme may be a proteolytic enzymeof plant origin, e.g., papain, bromelain, ficin, or of animal origin,e.g., trypsin and chymotrypsin, or of microbial origin, i.e., bacterial,yeast, or filamentous fungal. It is understood that any mixture ofvarious proteolytic enzyme may be applicable in the process of theinvention.

In another preferred embodiment, the other enzyme is a proteolyticenzyme such as a serine protease, a metalloprotease, or an aspartateprotease.

A sub-group of the serine proteases are commonly designated assubtilisins. A subtilisin is a serine protease produced by Gram-positivebacteria or fungi. The amino acid sequence of a number of subtilisinshave been determined, including at least six subtilisins from Bacillusstrains, namely, subtilisin 168, subtilisin BPN, subtilisin Carlsberg,subtilisin DY, subtilisin amylosacchariticus, and mesentericopeptidase,one subtilisin from an actinomycetales, thermitase fromThermoactinomyces vulgaris, and one fungal subtilisin, proteinase K fromTritirachium album. A further subgroup of the subtilisins, subtilases,has been recognised more recently. Subtilases are described as highlyalkaline subtilisins and comprise enzymes such as subtilisin PB92(MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, Novozymes A/S),and subtilisin 147 (ESPERASE®, Novozymes A/S).

A “subtilisin variant or mutated subtilisin protease” is defined hereinas a subtilisin that has been produced by an organism which isexpressing a mutant gene derived from a parent microorganism whichpossessed an original or parent gene and which produced a correspondingparent enzyme, the parent gene having been mutated in order to producethe mutant gene from which said mutated subtilisin protease is producedwhen expressed in a suitable host. These mentioned subtilisins andvariants thereof constitute a preferred class of proteases which areuseful in the method of the invention. An example of a useful subtilisinvariant is a variant of subtilisin 309 (SAVINASE®) wherein, in position195, glycine is substituted by phenylalanine (G195F or ¹⁹⁵Gly to¹⁹⁵Phe).

Commercially available proteases may be used in the methods of thepresent invention. Examples of such commercial proteases are ALCALASE®(produced by submerged fermentation of a strain of Bacilluslicheniformis), ESPERASE® (produced by submerged fermentation of analkalophilic species of Bacillus), RENNILASE® (produced by submergedfermentation of a non-pathogenic strain of Mucor miehel), SAVINASE®(produced by submerged fermentation of a genetically modified strain ofBacillus), e.g., the variants disclosed in the International PatentApplication published as WO 92/19729, and DURAZYM® (a protein-engineeredvariant of SAVINASE®), POLARZYME™, EVERLASE™. All the above-mentionedcommercial proteases are available from Novozymes A/S, DK-2880Bagsvaerd, Denmark.

Other preferred serine proteases are proteases from Aspergillus,Bacillus such as Bacillus alcalophilus, Bacillus cereus, Bacillusvulgatus, Bacillus mycoide, Rhizopus, and subtilins from Bacillus,especially proteases from the species Nocardiopsis such as Nocardiopsisnatto Nocardiopsis dassonvillei (see, WO 88/03947), especially proteasesfrom the species Nocardiopsis sp. NRRL 18262, and Nocardiopsisdassonvillei NRRL 18133. Yet other preferred proteases are the serineproteases from mutants of Bacillus subtilisins disclosed in theInternational Patent Application No. PCT/DK89/00002 and WO 91/00345, andthe proteases disclosed in EP 415 296.

Another preferred class of proteases is the metalloproteases ofmicrobial origin. Conventional fermented commercial metalloproteases maybe used in the methods of the present invention such as is NEUTRASE®(Zn) (produced by submerged fermentation of a strain of Bacillussubtilis), available from Novozymes A/S, DK-2880 Bagsvwerd, Denmark;BACTOSOL® WO and BACTOSOL® SI, available from Sandoz AG, Basle,Switzerland; TOYOZYME®, available from Toyo Boseki Co. Ltd., Japan; andPROTEINASE K® (produced by submerged fermentation of a strain ofBacillus sp. KSM-K16), available from Kao Corporation Ltd., Japan.

The protease may be used in a dosage of between 0.005 to 500 mg enzymeprotein per L biofilm control solution, preferably between 0.01 to 100mg enzyme protein per L biofilm control solution.

Lipases

In another preferred embodiment, the other enzyme is a lipase,especially a microbial lipase. As such, the lipase may be selected fromyeast, e.g., Candida; bacteria, e.g., Pseudomonas or Bacillus; orfilamentous fungi, e.g., Humicola or Rhizomucor. More specifically,suitable lipases may be the Rhizomucor miehei lipase (e.g., prepared asdescribed in EP 238 023), Thermomyces lanuginosa lipase e.g., preparedas described in EP 305 216, Humicola insolens lipase, Pseudomonasstutzeri lipase, Pseudomonas cepacia lipase, Candida antarctica lipase Aor B, or lipases from RGPL, Absidia blakesleena, Absidia corymbifera,Fusarium solani, Fusarium oxysporum, Penicillum cyclopium, Penicillumcrustosum, Penicillum expansum, Rhodotorula glutinis, Thiarosporellaphaseolina, Rhizopus microsporus, Sporobolomyces shibatanus,Aureobasidium pullulans, Hansenula anomala, Geotricum penicillatum,Lactobacillus curvatus, Brochothrix thermosohata, Coprinus cinerius,Trichoderma harzanium, Trichoderma reesei, Rhizopus japonicus, orPseudomonas plantari. Other examples of suitable lipases may be variantsof any one of the lipases mentioned above, e.g., as described in WO92/05249 or WO 93/11254.

Examples of commercially available lipases include: LIPOLASE™, LIPOLASEULTRA™, LIPOPRIME™, LIPEX™ from Novozymes, Denmark).

The lipase may be used in a dosage of between 0.005 to 500 mg enzymeprotein per L biofilm control solution, preferably between 0.01 to 100mg enzyme protein per L biofilm control solution.

Cellulases

In another preferred embodiment, the other enzyme is a cellulase orcellulolytic enzyme, which refers to an enzyme which catalyses thedegradation of cellulose to glucose, cellobiose, triose and othercellooligosaccharides. Preferably, the cellulase is an endoglucanase,more preferably a microbial endoglucanase, especially a bacterial orfungal endoglucanase. Examples of bacterial endoglucanases areendoglucanases obtained from or producible by bacteria from the group ofgenera consisting of Pseudomonas or Bacillus lautus.

The cellulase or endoglucanase may be an acid, neutral, or alkalinecellulase or endoglucanase, i.e., exhibiting maximum cellulolyticactivity in the acid, neutral or alkaline pH range, respectively.Accordingly, a useful cellulase or endoglucanase is an acid cellulase orendoglucanase, preferably a fungal acid cellulase or endoglucanase, morepreferably a fungal acid cellulase or endoglucanse enzyme withsubstantial cellulolytic activity under acidic conditions, which isobtained from or producible by fungi from the group consisting ofTrichoderma, Actinomyces, Myrothecium, Aspergillus, and Botrytis.

A preferred acid cellulase or endoglucanase is obtained from the groupconsisting of Aspergillus niger, Aspergillus oryzae, Botrytis cinerea,Myrothecium verrucaria, Trichoderma longibrachiatum, Trichoderma reesei,and Trichoderma viride.

Another useful cellulase or endoglucanase is a neutral or alkalinecellulase or endoglucanse, preferably a fungal neutral or alkalinecellulase or endoglucanse, more preferably a fungal alkaline cellulaseor endoglucanase with substantial cellulolytic activity under alkalineconditions, which is obtained from fungi selected from the groupconsisting of Acremonium, Aspergillus, Chaetomium, Cephalosporium,Fusarium, Gliocladium, Humicola, Irpex, Myceliophthora, Mycogone,Myrothecium, Papulospora, Penicillium, Scopulariopsis, Stachybotrys, andVerticillium.

A preferred alkaline cellulase or endoglucanase is obtained from thegroup consisting of Cephalosporium sp., Fusarium oxysporum, Humicolainsolens, or Myceliopthora thermnophila, or preferably from the groupconsisting of Cephalosporium sp., RYM-202, Fusarium oxysporum, DSM 2672,Humicola insolens, DSM 1800, or Myceliopthora thermophila, CBS 117.65.

In another preferred embodiment, the other enzyme is a xylanase such asan endo-1,3-beta-xylosidase (EC 3.2.1.32), xylan 1,4-beta-xylosidase (EC3.2.1.37), and alpha-L-arabinofuranosidase (EC 3.2.1.55). Preferably thexylanase is obtained from Aspergillus aculeatus (an enzyme exhibitingxylanase activity, which enzyme is immunologically reactive with anantibody raised against a purified xylanase derived from Aspergillusaculeatus CBS 101.43, see, for example, WO 94/21785); Aspergillus oryzae(see, for example, SU 4610007); Aureobasidium pullulans (see, forexample, EP 0 373 107 A2); Bacillus circulans (WO 91/18978); Bacilluspumilus (see, for example, WO 92/03540); Bacillus stearothermophilus(see, for example, WO 91/18976, WO 91/10724); Bacillus sp. AC13(especially the strain NCIMB 40482, see, for example, WO 94/01532);Humicola insolens (see, for example, WO 92/17573); Rhodothermus (see,for example, WO 93/08275); Streptomyces lividans (see, for example, WO93/03155); Streptomyces viridosporus (see, for example, EP 496 671 A);Bacillus licheniformis (see, for example, JP 9213868); Thermoascusaurantiacus (see, for example, U.S. Pat. No. 4,966,850); Trichodermalongibrachiatum and Chainia sp. (see, for example, EP 0 353 342 A1);Trichoderma harzianum and Trichoderma reseei (see, for example, U.S.Pat. No. 4,725,544); Thermomyces lanuginosus (see, for example, EP 0 456033 A2); Thermomonospora fusca (see, for example, EP 0 473 545 A2);Trichoderma longibrachiatum (see W. J. J. van den Tweel et al., Eds.,Stability of Enzymes, Proceedings of an International Symposium held inMaastrich, The Netherlands, 22-25 November 1992, Fisk, R. S. andSimpson, pp.323-328); Dictyoglomus (see, for example, WO 92/18612);Streptomyces (see, for example, U.S. Pat. No. 5,116,746); and/orThermotoga (see, for example, WO 93/1917). Other examples of suitablexylanases may be variants (derivatives or homologues) of any one of theabove-noted enzymes having xylanolytic activity.

Examples of commercially available cellulase containing productsinclude: NOVOZYM™ 342, CELLUZYME™, CAREZYME™, RENOZYME™ (all Novozymes,Denmark).

The cellulase may be used in a dosage of between 0.005 to 500 mg enzymeprotein per L biofilm control solution, preferably between 0.01 to 100mg enzyme protein per L biofilm control solution.

Pectinases

In another preferred embodiment, the other enzyme is a pectinase such asa polygalacturonase (EC 3.2.1.15), pectinesterase (EC 3.2.1.11), orpectin lyase (EC4.2.2.10). A suitable source organism for pectinases maybe Aspergillus niger.

In another preferred embodiment, the other enzyme in the alpha-amylasecomposition comprises a hydrolytic enzyme composition produced by astrain of the fungus Aspergillus aculeatus, preferably Aspergmlusaculeatus, CBS 101.43. It is known that this strain produces an enzymecomposition comprising pectinolytic and a range of hemicellulolyticenzyme activities.

Examples of commercially available cellulase containing productsinclude: BioPrep™, SCOURZYME™ and PECTAWASH™ (Novozymes, Denmark).

The pectinase may be used in a dosage of between 0.005 to 500 mg enzymeprotein per L biofilm control solution, preferably between 0.01 to 100mg enzyme protein per L biofilm control solution.

Oxidoreductase

In another embodiment of the invention the alpha-amylase is combinedwith an oxidoreductase, such as an oxidase, peroxidase, or laccase.

-   a) Laccases act on molecular oxygen and yield water (H₂O) without    any need for peroxide (e.g. H₂O₂),-   b) Oxidases act on molecular oxygen (O₂) and yield peroxide (H₂O₂),    and-   c) Peroxidases act on peroxide (e.g. H₂O₂) and yield water (H₂O).

Examples of laccases (E.C. 1.10.3.2) include laccases derived from astrain of Polyporus sp., in particular a strain of Polyporus pinsitus orPolyporus versicolor, or a strain of Myceliophthora sp., in particularM. thermophila, a strain of Scytalidium sp., in particular S.thermophilium, a strain of Rhizoctonia sp., in particular Rhizoctoniapraticola or Rhizoctonia solani, or a strain of a Rhus sp., inparticular Rhus vernicifera. The laccase may also be derived from afungus such as Collybia, Fomes, Lentinus, Pleurotus, Aspergillus,Neurospora, Podospora, Phlebia, e.g. P. radiata (WO 92/01046), Coriolussp., e.g., C. hirsitus (JP 2-238885), or Botrytis.

In specifically contemplated embodiments, the laccase may be selectedfrom the group consisting of: the Polyporus pinisitus laccase (alsocalled Trametes villosa laccase) described in WO 96/00290, theMyceliophthora thermophila laccase described in WO 95/33836, theScytalidium thermophilium laccase described in WO 95/33837, thePyricularia oryzae laccase which can be purchased from SIGMA under thetrade name SIGMA no. L5510, the Coprinus cinereus laccase described inWO 96/06930, and the Rhizoctonia solani laccase described WO 95/07988.

Examples of peroxidases (1.11.1.7) include peroxidases derived fromplants (e.g., horseradish peroxidase) or micro-organisms including fungiand bacteria, such as a strain of Coprinus sp., such as Coprinuscinereus or Coprinus macrorhizus, or bacteria such as Bacillus, such asBacillus pumilus.

In specifically contemplated embodiments, the peroxidase may be selectedfrom the group consisting of: the Coprinus cinereus IFO8371 peroxidaseor variants thereof described in WO 95/10602, and the haloperoxidaseoriginating from a strain of Curvularia verruculosa CBS 147.63 describedin WO 97/04102.

Contemplated oxidases include especially carbohydrate oxidases, whichare enzymes classified under EC 1.1.3. Carbohydrate oxidases includeglucose oxidase (E.C. 1.1.3.4), hexose oxidase (E.C. 1.1.3.5) xylitoloxidase, galactose oxidase (E.C. 1.1.3.9), pyranose oxidase (E.C.1.1.3.10), alcohol oxidase (E.C. 1.1.3.13).

Carbohydrate oxidases may be derived from any origin, including,bacterial, fungal, yeast or mammalian origin.

Examples of glucose oxidases include glucose oxidases derived fromAspergillus sp., such as a strain of Aspergillus niger, or from a strainof Cladosporium sp. in particular Cladosporium oxysporum, especially Cl.oxysporum CBS 163 described in WO 95/29996.

Examples of hexose oxidases include hexose oxidases produced by the redsea-weed Chondrus crispus (commonly known as Irish moss) (Sullivan andIkawa, (1973), Biochim. Biophys. Acts, 309, p. 11-22; Ikawa, (1982),Meth. in Enzymol. 89, carbohydrate metabolism part D, 145-149) thatoxidizes a broad spectrum of carbohydrates and the red sea-weedIridophycus flaccidum that produces easily extractable hexose oxidases,which oxidize several different mono- and disaccharides (Bean andHassid, (1956), J. Biol. Chem, 218, p. 425; Rand et al. (1972, J. ofFood Science 37, p. 698-710).

The oxidoreductase may be used in a dosage of between 0.005 to 500 mgenzyme protein per L biofilm control solution, preferably between 0.01to 100 mg enzyme protein per L biofilm control solution.

In a final aspect the invention relates to the use of a Bacillusalpha-amylase for preventing, removing, reducing, or disrupting biofilmformation on a surface. In a preferred embodiment the alpha-amylase is aBacillus alpha-amylase, preferably one mentioned above in the“Alpha-Amylase” section.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

Materials & Methods

Chemicals used as buffers and reagents were commercial products of atleast reagent grade.

Enzymes:

Alpha-amylase A is a variant alpha-amylase of the parent Bacillus sp.alpha-amylase disclosed as SEQ ID NO: 2 in WO 00/60060. The amino acidsequence of said alpha-amylase has the following six amino aciddeletions/substitutions:

D183*+G184*+R118K+N195F+R320K+R458K

The variant is also disclosed in WO 01/66712. The alkaline alpha-amylasewas produced in batch 03AGE014-4.

Alpha-amylase B is derived from a strain of Bacillus flavothermus and isdisclosed in SEQ ID NO: 4.

Alpha-amylase C is derived from a strain of Bacillus licheniformis andis shown as SEQ ID NO: 6 in WO 99/19467.

Protease E is a Bacillus clausii (old name: Bacillus lentus C360=NCIB10309) subtilisin having a M222S substitution covered by EP patent no.396,608-B1 (Available on request from Novozymes, Denmark).

Lipase A is a lipase variant derived from Humicola lanuginosa strain DSM4109 having the following mutations: T231R, N233R disclosed in U.S. Pat.No. 6,939,702-B (Available on request from Novozymes).

Cellulase A is a multi-component cellulase from Humicola insolens(Available on request from Novozymes, Denmark).

Bacterial strains.

-   Bacillus subtilis obtained from ATCC 10774.-   E. coli ATCC #11229 and ATCC #25922-   Biofilm medium. Tryptic Soy Broth (TSB, purchased from VWR, P/N    DF0370-07) medium was prepared according to the manufacturer's    instructions, then diluted to 5% with water. 2 ml of trace elements    were added per liter.-   Agar. Tryptic Soy Agar (TSA, purchased from VWR, P/N DF0369-17) was    used as per the manufacturer's directions.-   Trace element solution. Per liter: 1.5 g CaCl₂, 1.0 g FeSO₄7.H₂O,    0.35 g MnSO₄.2H₂O, 0.5 g, NaMoO₄.-   Stainless steel coupons. Stainless steel coupons No. 304 were    obtained from Metal Samples Company (Munford, Ala.).-   BiOLC Ion Chromatography System. The IC system consisted of the    following components:-   GP50 Gradient Pump (P/N 059493)-   ED50A Electrochemical Detector (P/N 059499)-   AS50 Temperature Controlled Autosampler (P/N 056565)-   Electrochemical Cell for Integrated Amperometry, complete with Gold    Electrode and Ag/AgCl Reference Electrode (P/N 060386)-   Chromelion Data Control Software CHM-1-IC (P/N 060930)-   CDC Biofilm Reactor. Purchased from Biosurface Technologies, Inc.    (P/N CBR 90-2) complete with polycarbonate coupons (24 for each    reactor, P/N RD 128-PC).-   Detergent Cleaner Base. Obtained from Weiman Products (IL, USA) as    Burnishine Me.—multiple enzyme detergent. The enzymes were denatured    prior to use by heating in a microwave on high setting for 1 minute.

Methods: Alpha-Amylase Activity (KNU)

The amylolytic activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e. at 37° C.±0.05; 0.0003 MCa²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylumsolubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Determination of Degree of Identity Between Two Sequences

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10, and gap length penaltyof 10. Pairwise alignment parameters were Ktuple=1, gap penalty=3,windows=5, and diago-nals=5.

EXAMPLES Example 1

Biofilm Removal Using Alpha-Amylase A and Al pha-Amylase C

Biofilm reactors consisted of a 400 ml beaker, a magnetic stirrer, and 2stainless steel coupons. The coupons are taped vertically to the sidesof the beaker so that the bottom edge of the coupon rested on the bottomof the beaker. A stir bar is added and the beakers are covered with acircle of aluminum foil and autoclaved. 200 ml of sterile biofilm mediumis added to each beaker. To prepare the inoculum, each bacterial strain(from Bacillus subtilis) is grown overnight at 28° C. on plate countagar. Using a sterile swab, each is suspended in sterile water to anOD₆₈₆ of 0.100 and then diluted additionally to 10⁻¹. Each assayconsisted of 4 control beakers without enzyme, 2 beakers with 50 mg ofenzyme protein per liter of solution, and 2 beakers with 100 mg ofenzyme protein per liter of solution. Beakers are first incubated at 37°C. overnight with stirring to grow the biofilm on the stainless steelcoupons. Following this incubation step, the enzymes are added in the 2dosages noted above as per the table below and incubated for anadditional 2 hours at 40° C. Thereafter, each beaker and stainless steelcoupon is rinsed carefully with sterile water, stained with crystalviolet, rinsed with steile water, the remaining biofilm solubilized withacetic acid, and the absorbance of an aliquot of each solution ismeasured at 600 nm using a spectrophotometer. The measured absorbancesof the solutions provide a direct indication of the amount of biofilmremaining on the stainless steel coupons. A low absorbance correspondsto a good enzyme effect and little remaining biofilm, whereas a highabsorbance corresponds to a poor (or lack thereof) enzyme effect andconsiderable remaining biofilm.

Sample # Enzyme Used Enzyme Protein Conc. 1 Alpha-amylase A 50 mg per L& 100 mg per L 2 Alpha-Amylase C 50 mg per L & 100 mg per L 3 No enzyme(control) NA

Example 2 Raw Starch Solubilization Using Alpha-Amylase A, B, and C

The rate at which various alpha-amylases solubilize raw, unhydratedwheat starch was measured. Alpha-Amylase A, B and C, respectively, wereused in the study.

Twenty five milliliters of a 1% raw wheat starch solution with pH 8 trisbuffer and 15° dH was poured into a tube with lid and placed in a 40° C.water bath. The starting level of “reducing ends” was measured prior toaddition of enzyme. The enzyme concentration used in the study was 3 mgenzyme protein per g raw wheat starch. One milliliter samples were takenout at different times. Twenty microliters of 1 M HCl was added prior toincubation at 99° C. for 10 minutes. The combination of acid and heatinactivates the amylase. Then 20 microL 1M NaOH was added to make surethe sample was no longer acidic. The sample was then diluted, incubatedwith color reagent (PHABH, potassium sodium tartrate, NaOH) at 95° C.for 10 minutes and finally centrifuged before measuring the OD at 410 nmon the supernatant. The control (100% hydrolyzed starch) was made byincubating a solution of 1% raw wheat starch in 1M HCl in an oven at110° C. for 4 hours. This treatment was used to calculate the maximumamount of glucose which could be produced per gram of the raw wheatstarch. This value was set to 100% in the graph shown in FIG. 1.

For Alpha-Amylase A and B it can be seen that the initial rate of rawwheat starch solubilization observed within the first 5 hours issignificantly more rapid than for Alpha-Amylase C. This resulted in agreater percentage of the starch being solubilized by the former twoalpha-amylases vs. for the latter over this time period.

Example 3

Biofilm Removal Using Alpha-Amylase A and C in Combination with ProteaseE and Detergent

A mono-component biofilm of Escherichia coli (ATCC #11229) is grown onpolycarbonate coupons in a pre-sterilized CDC biofilm reactor. At thestart of the experiment, cultures of E. coli were grown on tryptic soyagar (TSA) overnight at 37° C. The next morning, a single colony waspicked from the plate using a 1 microL sterile inoculation loop andadded to a solution of 40 g TSB/liter of water. This solution wasincubated at 37° C. overnight to grow up the culture. The following day,1 milliliter of this culture was added to 400 milliliters of minimalmedia (0.30 g TSB/liter sterile water) contained in the CDC biofilmreactor. The solution was slowly stirred at 130 rpm and grown for 2 daysat 22° C. in a non fed batch mode. After the 2 day growth period, thecoupon holder rods and coupons were removed from the reactor, rinsed insterile dilution water to remove planktonic cells, the coupons werecarefully removed from the rods and then incubated at 40° C. for 1 hourin the following solutions (30 milliliters each).

-   A: Detergent cleaner base alone, 0.21 g detergent in sterile water-   B: Detergent cleaner base, 0.21 g+0.51 mg enzyme protein Protease    E+0.06 mg enzyme protein Alpha-amylase A-   C: Detergent cleaner base, 0.21 g+0.51 mg enzyme protein Protease    E+0.16 mg enzyme protein Alpha-amylase C.

Following the incubation step, the coupons were removed. The solutionswere filtered through 0.45 □micro m Nylon syringe filters and theirsugar contents measured by Ion Chromatography. PA100 guard andanalytical columns (P/N 043055) were used for the separation. A mobilephase gradient between 60/40 deionized water/100 mM NaOH and 100% 100 mMNaOH/1M Sodium Acetate (exponential gradient started 10 minutes into theseparation and ended at 85 minutes) was used to effect the separation.FIG. 2 shows an overlay of the 3 chromatograms generated in thisexperiment. When Alpha-Amylase A is used, a significantly higher levelof low molecular weight sugars (glucose=glu, maltose=mal,maltotriose=DP3, maltotetraose=DP4, maltopentaose=DP5) were generatedversus with the detergent alone or with Alpha-Amylase C. This indicatedan increased level of biofilm removal through increased breakdown of theamylopectin exopolysaccharides produced by the Ecoli bacteria.

Example 4

Biofilm Removal Using Alpha-Amylase A and C in Combination with ProteaseE, Cellulase A, Lipase A and Detergent.

Two CDC biofilm reactors filted with polycarbonate coupons wereautoclaved and filled with sterile 1/10 strength tryptic soy broth (TSB,3 g/liter strength) and inoculated with 1 ml log phase culture ofEscherichia coli (ATCC # 25922). The initial cell count in the reactorsaveraged to 5×10⁸ cfu/mL. Both reactors were operated for 24 hours inbatch mode at 37° C. (no inflow or outflow). After this period,continuous flow of the 1/10 TSB was started at a flowrate of 12 ml/minat 37° C. The E. coli biofilm was grown for 4 days. After this time, 1rod from each reactor (labeled as reactor 1 or 2) was pulled and placedinto sterile glass beakers containing two hundred milliliters of thefollowing filter-sterilized solutions.

-   A: Detergent cleaner base alone, 1.4 g detergent in sterile water-   B: Detergent cleaner base, 1.4 g+3.4 mg enzyme protein Protease    E+0.48 mg enzyme protein Lipase A+0.23 mg enzyme protein Cellulase    A+0.40 mg enzyme protein Alpha-amylase A-   C: Detergent cleaner base, 1.4 g+3.4 mg enzyme protein Protease    E+0.48 mg enzyme protein Lipase A+0.23 mg enzyme protein Cellulase    A+0.40 mg enzyme protein Alpha-amylase C.

The solutions in each of the beakers were incubated at 40° C. withmoderate stirring for 30 minutes, after which each of the rods weregently rinsed in sterile water. Finally, E. coli was enumerated on 2 ofthe 3 coupons from each rod using tryptic soy agar (TSA). The averageplate count results obtained from the study were as follows:

Average log₁₀ cfu/cm² Treatment on coupons A 4 × 10⁷ B 3 × 10⁴ C 2 × 10⁵

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-22. (canceled)
 23. A method for preventing, removing, reducing ordisrupting biofilm present on a surface, comprising contacting thesurface with an alpha-amylase derived from a bacterium.
 24. The methodof claim 23, wherein the alpha-amylase is derived from a strain ofBacillus.
 25. The method of claim 24, wherein the alpha-amylase isderived from a strain of Bacillus sp. NCIB 12289, NCIB 12512, NCIB12513, DSM 9375, DSMZ 12649, KSM AP1378, KSM K36, or KSM K38.
 26. Themethod of claim 24, wherein the alpha-amylase has an amino acid sequenceof at least 60% identity to SEQ ID NO: 2, 4 or
 6. 27. The method ofclaim 24, wherein the alpha-amylase has the amino acid sequence shown inSEQ ID NO: 2, 4 or
 6. 28. The method of claim 24, wherein thealpha-amylase has a deletion in positions D183 and/or G184 (using SED IDNO; 2 for numbering).
 29. The method of claim 28, wherein thealpha-amylase further has one or more of the following substitutions,R118K, N195F, R320K, R458K (using SEQ ID NO: 2 for numbering).
 30. Themethod of claim 29, wherein the alpha-amylase has the followingmutations:Delta (D183+G184)+R118K+N195F+R320K+R458K (using SEQ ID NO: 2 fornumbering).
 31. The method of claim 24, wherein the alpha-amylase has asubstitution in position N195F (using SEQ ID NO: 2 for numbering). 32.The method of claim 23, wherein the alpha-amylase comprisesAsn-Gly-Trh-Met-Met-Gin-Tyr-Phe-Glu-Trp in its N-terminal amino acidregion.
 33. The method of claims 23, wherein the surface is contactedfor between 1 minute and 2 days.
 34. The method of claim 23, whichfurther comprises contacting the surface with a surfactant.
 35. Themethod of claim 23, wherein the alpha-amylase is used in a concentrationof between 0.005-500 mg enzyme protein.
 36. The method of claim 23,wherein the alpha-amylase has a percentage (%) of hydrolyzed starch thatis higher than 15 after 5 hours at 40° C., 3 mg enzyme protein per gstarch, pH 8.0.
 37. The method of claim 23, which further comprisescontacting the surface with one or more additional enzymes selected fromthe group consisting of an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase,oxidoreductases, pectinolytic enzyme, peptidoglutamnase, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonucdease,transglutaminase, or xylanase.
 38. The method of claim 23, which furthercomprises contacting the surface with one or more agents selected fromthe group consisting of dispersants, surfactants, anti-microbials, andbiocides.
 39. The method of claim 23, wherein the surface is a hard,soft, or porous surface.
 40. The method of claim 39, wherein the surfaceis a membrane.
 41. The method of claim 23, wherein the biofilm removalis done at a temperature between 10-70°C.